Seeing the invisible: How RNA extraction and library prep shape viral read recovery in RNA-seq

Next-generation sequencing (NGS) is emerging as a powerful tool for detecting adventitious viral agents in biologics, aligning with recommendations from ICH Q5A(R2). However, the performance of RNA-seq-based detection assays is highly dependent on upstream sample preparation steps and data analysis. In this study, we systematically evaluated five RNA extraction methods and seven RNA-seq library preparation protocols across multiple sample matrices spiked with model viruses. Our results demonstrate variability in viral RNA recovery depending on extraction chemistry, virus, and sample matrix type. Tri reagent-based methods excelled for certain viruses, while magnetic bead-based extraction yielded the highest total RNA in most matrices tested. Among RNA-seq library prep methods, rRNA depletion post-library construction combined with dual indexing improved viral signal detection and minimized artifacts such as index hopping. Finally, we implemented a custom bioinformatics pipeline to enhance sensitivity and minimize host-background interference. These findings highlight critical analytical variables that influence NGS assay performance and provide practical guidance for optimizing workflow components to support the use of NGS for adventitious agent detection in cell and gene therapy product development.